Gene regulatory network analysis predicts cooperating transcription factor regulons required for FLT3-ITD+ AML growth

SUMMARY Acute myeloid leukemia (AML) is a heterogeneous disease caused by different mutations. Previously, we showed that each mutational subtype develops its specific gene regulatory network (GRN) with transcription factors interacting within multiple gene modules, many of which are transcription factor genes themselves. Here, we hypothesize that highly connected nodes within such networks comprise crucial regulators of AML maintenance. We test this hypothesis using FLT3-ITD-mutated AML as a model and conduct an shRNA drop-out screen informed by this analysis. We show that AML-specific GRNs predict crucial regulatory modules required for AML growth. Furthermore, our work shows that all modules are highly connected and regulate each other. The careful multi-omic analysis of the role of one (RUNX1) module by shRNA and chemical inhibition shows that this transcription factor and its target genes stabilize the GRN of FLT3-ITD+ AML and that its removal leads to GRN collapse and cell death.

Genes with shRNA hits -1 hit / 2 hits / 3 hits Genes with shRNA hits -1 hit / 2 hits / 3 hits     Heatmap shows the gene modules associated with each gene (black = associated, white = not).Genes which were single hits in the screen in 1 or more samples are in bold, if there were multiple hits in 1 or more samples they are in italics.Genes not in the RUNX1, AP1, CEBP, EGR1, FOX, NF1 module are not included in this data.Hierarchical clustering was performed to group genes in similar modules.

Figure
Figure S1 (related to Figures 1&2).Design of shRNA screen targeting the FLT3-ITD GRN A: Overlap of shared edges in FLT3-ITD and FLT3-ITD/NPM1 AML-specific TF networks.B: Scheme of GRN comparison between nine patients.C: Combined gene regulatory network showing all edges that are found in any ITD/ITD-NPM1 specific GRN.The color of the edges Figure S1 (related to Figures 1&2).Design of shRNA screen targeting the FLT3-ITD GRN A: Overlap of shared edges in FLT3-ITD and FLT3-ITD/NPM1 AML-specific TF networks.B: Scheme of GRN comparison between nine patients.C: Combined gene regulatory network showing all edges that are found in any ITD/ITD-NPM1 specific GRN.The color of the edges Figure S1

Figure S2 (
Figure S2 (Related to Figure2): Screening results and manual validation of selected hits in FLT3-ITD cell lines and primary cells A: MOLM14 in vivo screen results showing genes with shRNAs lost in the population, genes with multiple shRNA hits are highlighted.B: Scatter plot showing log2 shRNA frequency in MOLM14 in vivo screen, with lost shRNAs highlighted.C: MV4-11 in vitro screen results showing genes with shRNAs lost in the population, genes with multiple shRNA hits are highlighted.D: Scatter plot showing log2 shRNA frequency in MV4-11 in vitro screen, with lost shRNAs highlighted.E-H validation of GRN targets in MV4-11 cell lines, performed in duplicate for each construct, p-values were calculated using Student's t-test.Constructs that did not show a decrease in protein expression after 72 h induction were excluded from the analysis.E: Colony formation assays of MV4-11 cells transduced with shRNA targeting NFIL3.Induction of the shRNA knockdown of NFIL3 by doxycycline caused a decrease in colony formation compared to uninduced cells.The Western blot below shows a decrease in NFIL3 protein expression after 72 h induction, with GAPDH included as a control.F: Colony formation assays of MV4-11 cells transduced with shRNA targeting RUNX1.Induction of the shRNA knockdown of RUNX1 by doxycycline caused a decrease in colony formation compared to uninduced cells.The Western blot below shows a decrease in RUNX1 protein expression after 72 h induction, with GAPDH included as a control.G: Colony formation assays of MV4-11 cells transduced with shRNA targeting EGR1.Induction of the shRNA knockdown of EGR1 by doxycycline caused a decrease in colony formation compared to uninduced cells.The Western blot below shows a decrease in EGR1 protein expression after 72 h induction, with GAPDH included as a control.Note that EGR1 and EGR3 shRNA images were spliced together (bar).H: Expression of a dominant negative CEBP in MV4-11 clones showed a decrease in colony forming ability after induction compared to uninduced cells and MV4-11s transduced with an empty vector control.I: Histograms of counts normalised to no dox control of shRNA mini-screen performed in primary FLT3-ITD PDX cells from ITD-15 with lentiviral vectors expressing DOX-inducible selected shRNAs as indicated.Experiments were performed in duplicate with several independent shRNAs.NTC: Non-targeted control.J: Dose response curves of cell lines treated with DUSP1/6 inhibitor BCI.FLT3-ITD cell lines show sensitivity to the inhibitor at a similar level to cell lines with MAPK activating mutations (Kasumi-1, P31/FUJ) whilst those without show marginally decreased sensitivity, although all AML cell lines respond to the inhibitor.Means calculated from n=3 are plotted with ± SEM and IC50 are shown below.

Figure S3 (
Figure S3 (related to figure 3) TF modules of upregulated genes in FLT3-ITD AML: A-F: AP-1, FOX, RUNX, NFI, C/EBP and EGR regulatory modules of FLT3-ITD AML specifically expressed genes as compared to PBSCs.Node colour indicates gene expression in FLT3-ITD+ AML samples (FPKM).Edges indicate an interaction between TF family and target genes, with the

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Figure S4 (related to Figure 4).TF perturbation experiments in primary cells

Figure S5 (
Figure S5 (related to Figure 5) RUNX1 plays a key role in the maintenance of the FLT3-ITD AML phenotype:

Figure S6 (
Figure S6 (Related to Figure 6): Perturbation of RUNX1 with CBFβi in FLT3-ITD primary cells A: Representative images from proximity ligation assay (PLA) showing a time course of dissociation of the CBFβ::RUNX1 complex in primary FLT3-ITD (ITD-14) AML cells after treatment with 10 μM CBFβi.The red signal shows interactions between CBFβ and RUNX1 counterstained with DAPI (blue).Images show these interactions at 4 different time points.The scatter plot shows the number of red foci counted per cell in triplicate at the four different time points.Scale bar = 10 μm.B: Colony forming ability of primary FLT3-ITD AML and healthy cells after treatment with CBFβi.Significant p-values are indicated on the graph and were calculated using Student's t-test.C: Density plot of ATAC-Seq analysis (red) of a second primary FLT3-ITD patient cells (ITD-13) with and without CBFβi ranked against each other according to fold-change with the indicated TF motifs (black) at the open chromatin sites and the logFC RNA expression of the genes associated to the peaks present plotted alongside.D: Unsupervised clustering of the fold change of gene expression in 3 different patients (ITD-12, ITD-13, ITD-14) after 24 h treatment with 10 μM CBFβi or 0.1% DMSO control.Left panel: FLT3-ITD AML specific genes.Right panel: FLT3-ITD AML specific genes in RUNX1 module.E: Genes up-regulated in the RNA-seq data in 2 or more of the CBFbi treated patients.Heatmap shows the gene modules associated with each gene (black = associated, white = not).Genes which were single hits in the screen in 1 or more samples are in bold, if there were multiple hits in 1 or more samples they are in italics.Genes not in the RUNX1, AP1, CEBP, EGR1, FOX, NF1 module are not included in this data.Hierarchical clustering was performed to group genes in similar modules.